CN114617903B - Composition for freeze-drying blood plasma and application thereof - Google Patents

Composition for freeze-drying blood plasma and application thereof Download PDF

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CN114617903B
CN114617903B CN202210253269.5A CN202210253269A CN114617903B CN 114617903 B CN114617903 B CN 114617903B CN 202210253269 A CN202210253269 A CN 202210253269A CN 114617903 B CN114617903 B CN 114617903B
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CN114617903A (en
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汪德清
樊斌
钟晓龙
陈兴慧
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First Medical Center of PLA General Hospital
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Abstract

The invention discloses a composition for freeze-drying blood plasma, which comprises a protective agent before freeze-drying and a pH regulator after freeze-drying. The invention optimizes the preparation process of freeze-dried blood plasma, such as the preparation temperature, the kind and concentration of protective agent and pH regulator, so that the retention rate of total protein, fibrinogen and most of blood coagulation factors in the freeze-dried blood plasma is maintained at a higher level; the freeze-dried blood plasma has good stability and safety, and has the advantages of long storage time, convenient transportation, and convenient use in emergency rescue and war.

Description

Composition for freeze-drying blood plasma and application thereof
Technical Field
The invention relates to the field of plasma products, in particular to a composition for freeze-drying plasma and application thereof.
Background
In view of the series of problems of fresh frozen plasma in terms of preservation (-20 ℃) and transportation (cold chain), the requirements of emergency rescue in special environments and remote areas cannot be met, and the freeze-dried plasma becomes an ideal substitute for fresh frozen plasma in recovery and treatment of traumatic shock. Compared with fresh frozen plasma, the basic components of the freeze-dried plasma are the same as the freeze-dried plasma (including total proteins, fibrinogen and most of blood coagulation factors after freeze-drying), the freeze-dried plasma can be stored below 10 ℃ and transported below 25 ℃, and huge low-temperature cold chain equipment is not needed. The freeze-dried plasma is quickly re-dissolved before use, and the fresh frozen plasma needs at least 30 minutes of thawing time, which greatly improves the treatment efficiency. In case of unavailable melted plasma, the plasma can be used for emergency rescue of major accidents or natural disasters.
However, how to protect the activity of proteins and coagulation factors in freeze-dried plasma and to stabilize the pH value are also urgent problems to be solved.
The preparation method of freeze-dried blood plasma disclosed in patent 01106637.7 is added with protective agent, and before freeze-drying, it is necessary to make low-temperature spin-freezing, and the recovery rate of fibrinogen and some main coagulation factors can be up to 65%.
The plasma lyophilization procedure disclosed in patent 02103960.7 is performed with low-temperature spin-freezing prior to lyophilization for a total period of up to 100-120 hours, and no description is given of plasma protein activity in the lyophilized plasma obtained by preparation.
The prior art cannot ensure that the active protein in the re-dissolved plasma has higher retention rate after freeze-drying, and simultaneously can enable the pH of the plasma to be adjusted to be in a proper range, so that the electrolyte and the blood gas of a recipient are ensured to be maintained in a stable state, and the preparation method of the freeze-dried plasma in the prior art generally needs to perform low-temperature spin-freezing before freeze-drying, however, the spin-freezing before freeze-drying increases the operation difficulty, and the foreign pathogen pollution is likely to be generated.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a plasma treatment method and application of a protective agent and/or a regulator in freeze-dried plasma.
The invention provides a plasma treatment method, which comprises a plasma freeze-drying treatment step and a plasma re-dissolving treatment step, wherein the plasma freeze-drying treatment step comprises adding a protective agent into plasma, the re-dissolving treatment step comprises mixing the freeze-dried plasma with a renaturation solution to obtain a mixed solution, the pH value of the mixed solution is regulated by the renaturation solution, the pH value of the mixed solution is regulated to 7.2-7.6 by the renaturation solution, and the renaturation solution comprises a pH regulator and pure water.
Further, the step of adjusting the pH value of the mixed solution by using the renaturation solution means that the pH value of the mixed solution is adjusted to 7.3-7.5, preferably 7.35-7.45.
Further, the protective agent is mannitol, the concentration of mannitol in the blood plasma is 0.02 g/mL-0.1 g/mL (such as 0.02g/mL、0.025g/mL、0.03g/mL、0.035g/mL、0.04g/mL、0.045g/mL、0.05g/mL、0.055g/mL、0.06g/mL、0.065g/mL、0.07g/mL、0.075g/mL、0.08g/mL、0.085g/mL、0.09g/mL、0.095g/mL、0.1g/mL), preferably, the concentration of mannitol in the blood plasma is 0.02 g/mL-0.06 g/mL, more preferably, the concentration of mannitol is 0.025 g/mL).
Further, the pH regulator is phosphate buffer Pair (PBS).
Further, the concentration of the renaturation solution is 32-38 mg/mL (such as 32mg/mL、32.5mg/mL、33mg/mL、33.5mg/mL、34mg/mL、34.5mg/mL、35mg/mL、35.5mg/mL、36mg/mL、36.5mg/mL、37mg/mL、37.5mg/mL、38mg/mL), is preferably 35.35 mg/mL).
Further, the Phosphate Buffered Saline (PBS) pair consists of the following components:
Sodium chloride, potassium chloride, disodium phosphate monobasic, and potassium phosphate monobasic.
Further, the concentration of sodium chloride in the renaturation solution is 25-30 mg/mL, the concentration of potassium chloride is 0.5-1.5 mg/mL, the concentration of disodium hydrogen phosphate is 2-8 mg/mL, the concentration of potassium dihydrogen phosphate is 0.5-1.5 mg/mL, preferably, the concentration of sodium chloride in the renaturation solution is 26-29 mg/mL, the concentration of potassium chloride is 0.5-1.0 mg/mL, the concentration of disodium hydrogen phosphate is 3-7 mg/mL, the concentration of potassium dihydrogen phosphate is 0.6-1.2 mg/mL, more preferably, the concentration of sodium chloride in the renaturation solution is 28.6mg/mL, the concentration of potassium chloride is 0.7mg/mL, the concentration of disodium hydrogen phosphate is 5.15mg/mL, and the concentration of potassium dihydrogen phosphate is 0.85mg/mL.
In one embodiment of the present invention, the renaturation solution may be prepared by: a phosphate buffer pair having a mass of 7.07g was added to 200ml of pure water to serve as a renaturation solution, and the 7.07g phosphate buffer pair was composed of 5.72g NaCl, 0.14g KCl, and 1.03g Na 2HPO4、0.17g KH2PO4.
Further, the plasma freeze-drying treatment step is preceded by a plasma pretreatment step, wherein the pretreatment step is as follows:
(1) Collecting whole blood and placing the whole blood in a blood bag;
(2) And (3) centrifuging the whole blood in the step (1) at a certain temperature to separate plasma from red blood cells in the blood and obtain plasma.
Further, the temperature in the step (2) is 0 to 8 ℃, preferably 2 to 5 ℃, more preferably 4 ℃.
Further, the blood bag contains a red blood cell preservation solution, wherein the red blood cell preservation solution comprises citric acid, sodium citrate, glucose, sodium dihydrogen phosphate, adenine, sodium chloride and mannitol.
Further, the concentration of citric acid in the erythrocyte preservation solution is 0.1-0.5 mg/mL (such as 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5 mg/mL), the concentration of sodium citrate is 1.0-2.0 mg/mL (such as 1.0mg/mL、1.1mg/mL、1.2mg/mL、1.3mg/mL、1.4mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL、2.0mg/mL)、 glucose concentration is 5.0-10.0 mg/mL (such as 5.0mg/mL、5.5mg/mL、6.0mg/mL、6.5mg/mL、7.0mg/mL、7.5mg/mL、8.0mg/mL、8.5mg/mL、9.0mg/mL、9.5mg/mL、10.0mg/mL)、 sodium dihydrogen phosphate concentration is 0.5-1.5 mg/mL) (such as 0.5mg/mL、0.55mg/mL、0.6mg/mL、0.65mg/mL、0.7mg/mL、0.75mg/mL、0.8mg/mL、0.85mg/mL、0.9mg/mL、0.95mg/mL、1.0mg/mL、1.05mg/mL、1.5mg/mL)、 adenine concentration is 0.05-0.5 mg/mL (such as 0.05mg/mL、0.06mg/mL、0.07mg/mL、0.08mg/mL、0.09mg/mL、0.10mg/mL、0.11mg/mL、0.12mg/mL、0.13mg/mL、0.14mg/mL、0.15mg/mL、0.16mg/mL、0.17mg/mL、0.18mg/mL、0.19mg/mL、0.20mg/mL、0.25mg/mL、0.30mg/mL、0.35mg/mL、0.40mg/mL、0.45mg/mL、0.5mg/mL)、 sodium chloride concentration is 3.0-6.0 mg/mL (such as 3.0mg/mL、3.5mg/mL、4.0mg/mL、4.5mg/mL、4.6mg/mL、4.7mg/mL、4.8mg/mL、4.9mg/mL、5.0mg/mL、5.1mg/mL、5.2mg/mL、5.3mg/mL、5.4mg/mL、5.5mg/mL、6.0mg/mL)、 mannitol concentration is 10.0-20.0 mg/mL) (such as 10.0mg/mL、11mg/mL、12mg/mL、13mg/mL、13.5mg/mL、14mg/mL、14.1mg/mL、14.2mg/mL、14.3mg/mL、14.4mg/mL、14.5mg/mL、14.6mg/mL、14.7mg/mL、14.8mg/mL、14.9mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL), preferably), the concentration of citric acid in the erythrocyte preservation solution is 0.2mg/mL, the concentration of citric acid is 1.5mg/mL, the concentration of glucose is 7.93mg/mL, the concentration of sodium dihydrogen phosphate is 0.94mg/mL, the concentration of sodium chloride is 14.57mg/mL, and the concentration of mannitol is 14.0 mg/mL).
Further, the freeze-drying treatment step further comprises the step of cooling the plasma treated by the protective agent under the atmospheric pressure according to the following cooling steps:
Firstly, cooling to-5 ℃ and maintaining for 20-40 min;
Then cooling to-10 to-30 ℃ and maintaining for 20-40 min;
Cooling to-60 to-50 ℃ and maintaining for 800-1000 min;
Then heating according to the following heating steps and simultaneously vacuumizing:
firstly, heating to-45 to-35 ℃ and maintaining for 400-600 min;
heating to-33 to-25 ℃ and maintaining for 900-1200 min;
then heating to-20 to-5 ℃ and maintaining for 1200-1800 min;
heating to-3 deg.c and maintaining for 1800-2200 min;
heating to 5-12 deg.c for 800-1200 min;
heating to 13-18 ℃ and maintaining for 1300-1700 min;
Heating to 19-25 ℃ and maintaining for 800-1200 min;
The vacuum degree after vacuumizing is 0-5 Pa (such as 0Pa, 1Pa, 2Pa, 3Pa, 4Pa, 5 Pa),
Preferably, the freeze-drying treatment step further comprises the step of cooling the plasma treated with the protective agent at atmospheric pressure first according to the following cooling step:
Firstly, cooling to 0 ℃ and maintaining for 30min;
then cooling to-20 ℃ and maintaining for 30min;
cooling to-55deg.C, and maintaining for 900min;
Then heating according to the following heating steps and simultaneously vacuumizing:
Firstly, heating to-40 ℃ and maintaining for 500min;
heating to-30deg.C and maintaining for 1000min;
Heating to-10deg.C and maintaining for 1500min;
heating to 0deg.C and maintaining for 2000min;
heating to 10deg.C and maintaining for 1000min;
Heating to 15 ℃ and maintaining for 1500min;
Heating to 20deg.C and maintaining for 1000min;
The vacuum degree after vacuumizing is 0-3 Pa.
In a second aspect, the invention provides the use of a protective agent and/or a pH adjuster in freeze-dried plasma, wherein the protective agent is mannitol, the adjuster is phosphate buffer Pair (PBS), the protective agent and/or the pH adjuster can enable the retention rate of Total Protein (TP) in the freeze-dried plasma to be more than 60g/L (such as 60g/L, 61g/L, 62g/L, 63g/L, 64g/L, 64.5g/L, 65g/L, 66g/L, 67g/L, 68g/L, 69g/L, 70 g/L), the retention rate of Fibrinogen (FIB) in the freeze-dried blood plasma is more than 2.8g/L (such as 2.8g/L, 2.85g/L, 2.9g/L, 2.95g/L, 2.99g/L and 3.0 g/L), the retention rate of coagulation factor V (FVV) in the freeze-dried blood plasma is more than 88 percent (such as 88 percent, 88.5 percent, 89 percent, 89.5 percent, 89.7 percent, 90 percent, 90.5 percent, 91 percent, 91.5 percent and 92 percent), and the retention rate of coagulation Factor VIII (FVIII) in the freeze-dried blood plasma is more than 82 percent (such as 82 percent, 82.5 percent, 83 percent, 83.4 percent, 83.5 percent and 84 percent).
According to the invention, a proper amount of protective agent is added in the preparation process of freeze-dried plasma, so that the stability of proteins and coagulation factors in the plasma during freeze-drying and storage is improved; the pH regulator is also used as renaturation liquid to regulate the pH value in the process of re-dissolving the freeze-dried plasma, so that the problem of high plasma pH caused by escape of carbon dioxide gas from the plasma in the freeze-drying process of the freeze-dried plasma is effectively solved; the invention optimizes the preparation process of freeze-dried blood plasma, such as the preparation temperature, the kind and concentration of protective agent and pH regulator, so that the retention rate of total protein, fibrinogen and most of blood coagulation factors in the freeze-dried blood plasma is maintained at a higher level; the freeze-dried blood plasma has good stability and safety, and has the advantages of long storage time, convenient transportation and convenient use for combat readiness.
Drawings
FIG. 1 shows the protective effect of glycine at various concentrations on lyophilized plasma proteins.
FIG. 2 shows the protective effect of mannitol on lyophilized plasma proteins at various concentrations.
FIG. 3 is a graph showing the effect of different quality pH modifiers on lyophilized plasma pH.
Detailed Description
In order that the technical content of the present invention may be more clearly understood, the following detailed description of the embodiments is given only for better understanding of the content of the present invention and is not intended to limit the scope of the present invention.
The concentrations of mannitol D-M (macklin) described in the examples below, such as 1%, 2.5%, 5%, etc., are correspondingly specified as 1g, 2.5g, 5g, i.e., 0.01g/mL, 0.025g/mL, 0.05g/mL mannitol per 100mL of plasma.
Example 1
The invention utilizes a Pilot3-6 freeze dryer (Bo Yi kang laboratory instrument, beijing) to develop Pilot-scale plasma freeze-drying key technical research, and through continuously adjusting the freeze-drying process and a large amount of experimental research, the freeze-drying process of 200ml of freeze-dried plasma per bottle is finally determined as shown in Table 1:
TABLE 1
The plasma treated by the protective agent is firstly cooled under the atmospheric pressure according to the following cooling steps:
Firstly, cooling to 0 ℃ and maintaining for 30min;
then cooling to-20 ℃ and maintaining for 30min;
cooling to-55deg.C, and maintaining for 900min;
Then heating according to the following heating steps and simultaneously vacuumizing:
Firstly, heating to-40 ℃ and maintaining for 500min;
heating to-30deg.C and maintaining for 1000min;
Heating to-10deg.C and maintaining for 1500min;
heating to 0deg.C and maintaining for 2000min;
heating to 10deg.C and maintaining for 1000min;
Heating to 15 ℃ and maintaining for 1500min;
the temperature was raised to 20℃again and maintained for 1000min.
Under the freeze-drying process, the applicant adopts glycine (sigma) (0, 15 and 30 mM) with different concentrations as a protective agent to study the protective effect of freeze-dried plasma protein, and as shown in figure 1, the applicant finds that three different concentrations of glycine have no statistical significance (p > 0.05) on the differences of the protective effects of fibrinogen (Fib), blood coagulation Factor V (FV) and blood coagulation Factor VIII (FVIII).
The applicant continued to study the protective effect of lyophilized plasma proteins under the above lyophilization process conditions using mannitol D-M (macklin) (2.5%, 5%) at different concentrations, and as shown in fig. 2, the applicant found that the difference in the retention rate of fviii in the 5% D-M group was statistically significant (p < 0.05), indicating that 5% D-M had a protective effect on fviii during plasma lyophilization.
The applicant of pH adjusted the pH of the plasma after lyophilization by adding a pH adjuster to the renaturation solution in advance.
The applicant selects citric acid (sigma), hydrochloric acid (sigma), ascorbic acid (sigma) and phosphate buffer pair PBS (biosharp) as alternative pH regulator in sequence, and experimental researches show that citric acid, hydrochloric acid and ascorbic acid can lead plasma proteins to deform and separate out to form flocculent precipitate, so that the risk of thrombus formation of an infuser is greatly increased while the retention rate of the plasma proteins is influenced, and the phosphate buffer pair does not occur, so that the phosphate buffer pair is finally selected as the pH regulator.
The applicant studied the effect of different amounts of pH adjuster added on the pH of plasma after lyophilization, and set four experimental groups (control: 0g, groupA:6.06g, groupB:7.07g, groupC:8.08 g) to which different amounts of pH adjuster were added, and as a result, as shown in FIG. 3, group groupB of plasma pH was adjusted to a normal range (7.35-7.45), and thus, it was finally confirmed that 7.07g of phosphate buffer pair was added to 200ml of renaturation pure water as a renaturation liquid for adjusting the pH of plasma after lyophilization to a normal level.
To study the effect of possible interactions between lyoprotectant and pH adjuster on active substances during plasma lyophilization, applicant devised three influencing factors (preparation temperature, mannitol concentration, phosphate buffer versus mass) for orthogonal design, observing the effect of each factor on plasma active protein retention after lyophilization, each factor set three levels: temperature (4 ℃, 22 ℃, 37 ℃), mannitol concentration (1%, 2.5%, 5%), phosphate buffer pair mass (3.535 g, 7.07g, 10.605 g), total Protein (TP), fibrinogen (FIB), factor V (FV), factor VIII (FVIII) retention in the lyophilized plasma were measured, 3X3 orthogonal design was performed using orthogonal Table L9 (3 4), and the optimal production protocol was determined. Tables 2 to 5 show the effect of three factors on Total Protein (TP) in plasma;
TABLE 2
A. Determination coefficient=.800 (correction determination coefficient=.201)
TABLE 3 Table 3
TABLE 4 Table 4
TABLE 5
Tables 6-9 show the effect of three factors on Fibrinogen (FIB);
TABLE 6
TABLE 7
TABLE 8
TABLE 9
Tables 10 to 13 show the effects of three factors on coagulation Factor V (FV);
table 10
A. determination coefficient=. 960 (correction determination coefficient=. 839)
TABLE 11
Table 12
TABLE 13
Tables 14-17 show the effect of three factors on Factor VIII (FVIII).
TABLE 14
A. determination coefficient=.912 (correction determination coefficient=.648)
TABLE 15
Table 16
TABLE 17
The results of the orthogonal test are shown in Table 18:
TABLE 18 results of orthogonal experiments
From the point of view of the preparation process, the applicant has found an effect on TP, a > B > C, the optimum level being A1B2C2; the influence on the FIB, A > B > C, and the optimal level is A1B2C3; the effect on coagulation Factor V (FV), A > C > B, with optimal levels of A1B2C1; the effect on blood coagulation Factor VIII (FVIII), A > C > B, with optimal levels of A1B2C2.
From the standpoint of the preparation process, the applicant has chosen the combination with the highest retention of active protein as the final pilot production standard, and therefore has chosen group A1B2C2 as the final production standard, i.e. plasma is prepared at 4 ℃, added with 2.5% d-M and lyophilized; 200ml of purified water was added with 7.07g of phosphate buffer pair as a renaturation solution.

Claims (5)

1. The plasma treatment method is characterized by comprising a plasma freeze-drying treatment step and a freeze-drying plasma re-dissolving treatment step, wherein the plasma freeze-drying treatment step comprises the step of adding a protective agent into plasma, the re-dissolving treatment step comprises the step of mixing the freeze-drying plasma with a renaturation solution to obtain a mixed solution, and the pH value of the mixed solution is regulated by the renaturation solution, and the renaturation solution comprises a pH regulator and pure water;
The protective agent is mannitol;
The concentration of mannitol in the plasma is 0.02 g/mL-0.06 g/mL;
the freeze-drying treatment step further comprises the step of firstly cooling the plasma treated by the protective agent at the atmospheric pressure according to the following cooling steps:
Firstly, cooling to-5 ℃ and maintaining for 20-40 min;
then cooling to-10 to-30 ℃ and maintaining for 20-40 min;
cooling to-60 to-50 ℃ and maintaining for 800-1000 min;
Then heating according to the following heating steps and simultaneously vacuumizing:
firstly, heating to-45 to-35 ℃ and maintaining for 400-600 min;
heating to-33 to-25 ℃ and maintaining for 900-1200 min;
heating to-20 to-5 ℃ and maintaining for 1200-180min;
heating to-3~3 ℃ and maintaining 1800-220min;
Heating to 5-12 ℃ and maintaining for 800-1200 min;
heating to 13-18 ℃ and maintaining 1300-1700 min;
Heating to 19-25 ℃ and maintaining for 800-1200 min;
The vacuum degree after vacuumizing is 0-5 Pa;
The pH regulator consists of the following components: sodium chloride, potassium chloride, disodium phosphate monobasic, potassium phosphate monobasic;
the concentration of disodium dihydrogen phosphate in the renaturation solution is 3-7 mg/mL, and the concentration of monopotassium phosphate is 0.6-1.2 mg/mL;
The plasma freeze-drying treatment step is preceded by a plasma pretreatment step, wherein the pretreatment step is as follows:
(1) Collecting whole blood and placing the whole blood in a blood bag;
(2) Centrifuging the whole blood in the step (1) at a certain temperature to separate plasma from red blood cells in the blood to obtain plasma; the temperature in the step (2) is 2-5 ℃.
2. A method of treating plasma according to claim 1, wherein the mannitol is present in the plasma at a concentration of 0.025g/mL.
3. The method for treating plasma according to claim 1, wherein the concentration of disodium phosphate contained in the renaturation solution is 5.15 mg/mL and the concentration of potassium dihydrogen phosphate is 0.85 mg/mL.
4. The method of claim 1, wherein the lyophilizing step further comprises first cooling the plasma treated with the protective agent at atmospheric pressure according to the following cooling steps:
Firstly, cooling to 0 ℃ and maintaining for 30min;
then cooling to-20 ℃ and maintaining for 30min;
cooling to-55deg.C, and maintaining for 900min;
Then heating according to the following heating steps and simultaneously vacuumizing:
Firstly, heating to-40 ℃ and maintaining for 500min;
heating to-30deg.C and maintaining for 1000min;
Heating to-10deg.C and maintaining for 1500min;
heating to 0deg.C and maintaining for 2000min;
heating to 10deg.C and maintaining for 1000min;
Heating to 15 ℃ and maintaining for 1500min;
Heating to 20deg.C and maintaining for 1000min;
The vacuum degree after vacuumizing is 0-3 Pa.
5. Use of a protecting agent and a pH adjusting agent for increasing the retention of total protein and fibrinogen in plasma after lyophilization, said protecting agent being mannitol, said pH adjusting agent consisting of: 5.72g of sodium chloride, 0.14g of potassium chloride, 1.03g of disodium hydrogen phosphate and 0.17g of potassium dihydrogen phosphate, wherein the concentration of mannitol in plasma is 0.025g/mL, the plasma is prepared at 4 ℃, and the mannitol is added with 0.025g/mL and then freeze-dried; 200ml of pure water is added into the pH regulator to be used as renaturation liquid; the protective agent and the pH regulator enable the retention rate of total protein in the freeze-dried blood plasma to be more than 60g/L, enable the retention rate of fibrinogen in the freeze-dried blood plasma to be more than 2.8g/L, enable the retention rate of blood coagulation factor V in the freeze-dried blood plasma to be more than 88%, and enable the retention rate of blood coagulation factor VIII in the freeze-dried blood plasma to be more than 82%.
CN202210253269.5A 2022-03-15 2022-03-15 Composition for freeze-drying blood plasma and application thereof Active CN114617903B (en)

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